Optical scanning has provided a rapid, sensitive method for the analysis of a variety of targets. Optical analysis has a variety of features advantageous to current analytical requirements. These include:
1. Rapid scanning capability. An illumination beam may be optically moved in a line scan across a sample. As the beam moves across a layer on the analyzed substrate, the excited fluorescence from the sample is measured. This allows optical scanning systems to image discrete illumination areas in a rapid scan.
2. High sensitivity. The use of fluorescent detection provides a sensitive means of detecting a variety of targets. A fluorescent or other optically detectable dye may be conjugated to a variety of binding or labeling agents. The use of labels with specific optical signals provides for discrimination of the target both in vitro and in vivo. Conjugation of the optical label to a binding agent allows for optical detection with the selected specificity of the binding agent.
3. Imaging versatility. The use of optically detectable labels may be adapted to a number of different imaging formats. Optical detection may be employed in bioarrays, where binding to a spot on a surface is indicated by an optical signal. Alternatively, a binding agent may be used in a solution for binding to a solid substrate or cell.
4. Processing throughput. Optical scanning interrogates a discrete area of a substrate in a rapid scan. Such scanning is adaptable to multiplexing, with use of different dyes, arrays of reaction containers (e.g. wells on a multi-well plate), and pooling of samples to be analyzed all available as means to increase sample processing throughput. Automation of placement of analytical substrate on a stage for optical interrogation allows further increase in the sample processing, enabling automated high throughput assays.
One system for optical sample scanning is described in U.S. Pat. Nos. 5,547,849 and 5,556,764. In this system, a laser illumination beam is reflected by a beam splitter, and focused by an objective lens onto a sample container or substrate. Excited fluorescent light is gathered by the objective lens (which also acts as a light collector) and transmitted as a retrobeam to the beam splitter. The beam splitter is designed to reflect the excitation beam while transmitting the collected emission light. The beam passes through the beam splitter and is directed through a focal lens, which focuses the light through an aperture of a spatial filter and onto detection optics. The spatial filter acts to block emission light that originates from outside an interrogated depth of field from reaching the detection optics. The objective, spatial filter and illumination focus optics act in conjunction to limit the depth of field to a narrow plane.
The focal optics focus the illumination into a beam spot that concentrates the illumination energy into a selected depth. The objective will collect and collimate light originating from this depth. Out of focus emission light will not be focused through the aperture of the spatial filter and onto the detectors. In contrast to confocal imaging that images a depth of field of under 1 um, the depth of field of the described system is between 25–250 um. This provides a “virtual capillary” from which fluorescence may be detected.
This system provides a number of advantages for imaging. First, the limitation of detection to a thin detection depth allows for homogenous assay (i.e. no separation step). A sample mixture may contain a fluorescent binding agent that is present in an assay mixture both bound to a discrete target (e.g. a cell or solid substrate) and is also present free in solution. Because detection is limited to a narrow depth of field, the unbound optically detectable binding agent is detected only as a background signal. The greater concentrations of binding agent present at target binding sites produce a sufficient optical signal to allow detection over a background signal. The ability to perform a homogenous assay increases sample processing throughput by avoiding time consuming washing steps. In addition, possible error or cross contamination from washing steps is eliminated. Also contact with sample is minimized. This reduces risks from infectious or toxic samples.
A second advantage is versatility of the system. Although the system in the patents is described as scanning a capillary of limited depth, the described system may be adapted for optical analysis in a limited depth of a variety of sample containers or substrates.
A third advantage of the system is simplified alignment. When the illumination beam is in focus on its target, the resulting fluorescence, which originates from the same focal spot, is automatically in focus. The objective that focuses the illumination beam also acts as the light collector. Illumination and light collection optical alignment are never a problem because a single element serves a dual function of both illumination focus and emission light collection. This design also minimizes the space required for the optics and reduces the required number of optical elements, reducing system costs and assembly difficulty.
Despite these advantages, the described optical configuration presents certain limitations. In the present configuration a number of optical elements are common to both the illumination beam path and the detection (retrobeam) path. Thus eventually the illumination light must be optically separated from the fluorescent emission to allow detection. This may be achieved using a beam splitter, such as a coated dichroic mirror that reflects the excitation beam to the objective and transmits the excitation retrobeam to the detection optics. This requires design of this element to be compatible with only specific illumination sources and excitation wavelengths.
In certain applications it may be necessary to use different combinations of excitation wavelengths and different combination of dyes producing different emission wavelengths. However the beam splitter must be selected for a specific excitation and emission combination. To use different filters would require a very precise filter wheel. Because the alignment of this element is critical for proper targeting of the illumination beam, movable filters are technically impractical.
Alternatively, a dot mirror having a reflective dot surrounded by a transmissive element could be used. The illumination laser beam is much smaller than the diameter of the emitted fluorescence retrobeam. The illumination beam would thus be reflected while the outer annulus of the retrobeam is transmitted through the transmissive elements to the detection agents. In this design, multiple illumination and emission wavelengths are accommodated in a design that is intrinsically compatible with a broad wavelength range for both illumination and light collection. A broad spectrum of illumination wavelengths would be reflected by the central reflective element and a broad spectrum of emission wavelengths transmitted through the transparent annular disc.
However certain drawbacks to this system design are also expected. First, part of the retrobeam is blocked by the reflective center reflective mirror, resulting in a loss of signal. Because the energy of the signal is concentrated at the center, this loss would be significant. In addition, the objective must be broadly achromatic to allow for it to both focus the illumination beam and collect and collumnate the retrobeam. This creates design difficulties to produce an objective lens which is both efficient in light collection while also being broadly achromatic to allow both focus of the illumination light and collection of the emission light.
It is the object of the present invention to provide a photon efficient optical scanner which illuminates a sample and collects light from a large numerical aperture. It is a further object to provide a scanner that may be used with a variety of different illumination and emission wavelengths without changing optical elements. It is a further object of the invention to provide a scanner which could be adapted to scan a variety of sample media, including solid substrate surfaces, wells of a multiwell plate, capillaries, or other sample containers. It is a further object of the invention to describe a scanner that scans in a limited depth of field (allowing for homogenous assay of discrete targets). This scanner should be compact, versatile, allow for high speed scanning, and be adaptable to automation.